See instmctions): In this continuation application/progress report for an R37 MERIT Award, the progress on the two major specific aims is reviewed. The overall goal of the work is to use HIV-1 sequence diversity to examine the biological determinants of transmission and pathogenesis. These studies will inform our understanding of the transmission and disease processes. The first aim covering the study of samples from an HIV-1 vertical transmission cohort Is nearing completion. A major feature of the remaining work is to create molecular clones of the env genes from maternal and infant viral populations to test the rote of autologous neutralizing antibodies in vertical transmission. This work will test the hypothesis that a neutralization escape variant is responsible for vertical transmission. In addition, these studies will reveal the complexity of the virus in vertical transmission and provide reagents to study other biological properties of the transmitted virus. The second aim on horizontal transmission is still awaiting the availability of samples from people in acute infection from southem Braal. Given progress in the field this aim is now refined to focus on the question of the relative fitness of subtype B and subtype C viruses Involved in acute infection and coclrculating in the same population. This analysis is designed to address the question of whether differences in relative viral fitness play a role in the subtype B and C epidemics. Studies in viral diversity are in part framed by the technology available to study that diversity. Two important and complementary technologies for studying diversity are the heteroduplex tracking assay (HTA) and the single genome amplification (SGA) approach. The new technology of deep sequencing has not been approriately applied to the study of viral diversity due to intrinsic problems of PCR resampling. A new strategy will be applied to overcome this problem to allow the accurate use of this new technology to study viral diversity using HIV as the test case This will provide a third complementary approach to the study of viral diversity. The application of these strategies in ways that could inform clinical care will also be explored. Finally, collaborative opportunities have been developed to study viral evolution in a humanized mouse model and under the influence of AP0BEC3G/F and will be continued. RELEVANCE (See Instmctions): HIV sequence diversity is a rich source of biological infonnation about the selective pressures placed on the virus and changes in virus-host interactions. We are using viral sequence diversity to explore questions important for vertical and horizontal transmission and the evolution of pathogenic variants after transmission. In addition, we continue to develop new technologies to derive more information from complex viral populations. These efforts shed light on important virologic features of transmission and evolution. PROJECT/PERFOI^MANCE SiTE(S) (if addifional space is needed, use Project/Perfonnance Site Fonnat Page)